One of our main focus areas over the years is muscle development. In mice, striated muscle development requires the function of the MRFs, a set of four closely related factors (MyoD, Myf-5, MRF-4, Myogenin) that regulated muscle specification and differentiation. C. elegans has a single MRF-related factor called CeMyoD (HLH-1). One of the puzzling aspects of C. elegans myogenesis was the observation that muscles could be formed in the absence of CeMyoD, as demonstrated in mutant animals. To further define the exact role of this transcription factor, we ectopically expressed HLH-1/MyoD in the early nematode embryo and found that it was sufficient to convert all early blastomeres to a muscle-like fate. The sufficiency of HLH-1/MyoD alone to direct cells into the muscle lineage illustrated its potency and revealed a level of evolutionary conservation in function that had not previously been appreciated. Moreover, these experiments revealed a remarkable degree of plasticity of the early embryonic cells to be reprogrammed with regards to cell fate choice. Using genetic deletion alleles of three genes, we showed that muscle could form when at least one of these factors was present. However, elimination of all three gene products blocked muscle differentiation in the embryo. These results defined this trio of transcription factors as the key regulators of C. elegans muscle, explaining why muscle could form in the absence of HLH-1/MyoD. This past year, we have focused on the factors regulating anterior mesoderm specification and bodywall muscle development. Captializing on the defined cell lineage of C. elegans, we have defined a hierarchy of transcription factors involved regulating muscle development at the single cell level. This has allowed us to tease apart multiple pathways all leading to the eventual activation of HLH-1/MyoD and UNC-120/SRF, the common set of drivers for bodywall muscle fate specification and differentiation. Our results also revealed an unexpected evolutionary conservation of pathway components with anterior muscle development in vertebrates. We also completed our study of the Forkhead transcription factor UNC-130 to understand its transcriptional regulation and its action in promoting body wall muscle polarity cues. We have identified widespread, cis-acting elements in the unc-130 promoter that function to positively regulate ventral body wall muscle expression and negatively regulate dorsal body wall muscle expression. We have defined the temporal distribution of UNC-130 protein in body wall muscle cells during embryogenesis, demonstrated that this pattern is required to establish the dorsal-ventral polarity of UNC-129/TGF-beta, and shown that UNC-130 is not required post-embryonically to maintain the asymmetry of body wall muscle unc-129 expression. Finally, we have tested the impact of the depletion of a variety of transcription factors, repressors, and signaling molecules to identify additional regulators of body wall muscle UNC-130 polarity. Our results confirmed and extended earlier studies to clarify the mechanisms by which UNC-130 is controlled and affects the pattern of unc-129 expression in body wall muscle. In on-going collaborations with Dr. Eisenmann (Univ of MD, Baltimore County) we have explored development of the outer layer of cells of the animal. The development of the single cell layer skin or hypodermis of Caenorhabditis elegans is an excellent model for understanding cell fate specification and differentiation. Early in C. elegans embryogenesis, six rows of hypodermal cells adopt dorsal, lateral or ventral fates that go on to display distinct behaviors during larval life. Several transcription factors are known that function in specifying these major hypodermal cell fates, but our knowledge of the specification of these cell types is sparse, particularly in the case of the ventral hypodermal cells, which become Vulval Precursor Cells and form the vulval opening in response to extracellular signals. Previously, the gene pvl-4 was identified by the Eisenmann lab in a screen for mutants with defects in vulval development. We found by whole genome sequencing that pvl-4 is the Paired-box gene pax-3, which encodes the sole PAX-3 transcription factor homolog in C. elegans. pax-3 mutants show embryonic and larval lethality, and body morphology abnormalities indicative of hypodermal cell defects. We report that pax-3 is expressed in ventral P cells and their descendants during embryogenesis and early larval stages, and that in pax-3 reduction-of-function animals the ventral P cells undergo a cell fate transformation and express several markers of the lateral seam cell fate. Furthermore, forced expression of pax-3 in the lateral hypodermal cells causes them to lose expression of seam cell markers. We propose that pax-3 functions in the ventral hypodermal cells to prevent these cells from adopting the lateral seam cell fate. pax-3 represents the first gene required for specification solely of the ventral hypodermal fate in C. elegans providing insights into cell type diversification.